HIV envelope glycoprotein gp120 contains epitopes which are targeted by broadly cross- reactive neutralizing antibodies in humans and which depend on the native protein conformation. Based on mapping these epitopes on the three dimensional structure of gp120, we have found a distant site, where foreign protein sequences can be inserted without disrupting the native structure of the neutralizing site. By inserting a potent immunogen at this site, we could enhance the intrinsic vaccine potency of gp120, while retaining important conformational sites needed to elicit these antibodies. Two types of carrier protein have been inserted at this site, hepatitis B surface antigen (HBsAg) and hepatitis B core antigen. The resulting protein hybrids should combine the potent immunogenicity of the carrier protein with the native epitopes of gp120. Hybrid proteins were expressed in a recombinant vaccinia vector and showed normal glycosylation, dual antigenicity, and high affinity binding of CD4, indicating native folding at the CD4 binding site. Although native core antigen forms multimeric particles, few of the core/gp120 hybrids were able to assemble. One construct out of many tested gave particles, but in low yield. In contrast, the surface antigen/gp120 hybrid assembled particles with high efficiency. Unlike the rigid icosahedral structure of assembled core antigen, surface antigen assembles into flexible lipoprotein particles, which may accept surface antigen/gp120 hybrids more readily. These particles showed dual antigenicity and native folding of the CD4 binding site on gp120. They banded at a density of 1.17- 1.20 g/ml in sucrose, indicating a lipoprotein mix similar to native HBsAg particles. EM shows particles a little larger than the ones formed by surface antigen alone. These particles probably represent a regular array of about 200 gp120 molecules at the lipid/water interface. The only comparable assembly of gp120 is found on the surface of HIV virions. Assembly of gp120 into virus-like particles has been a long-term goal of this laboratory. The particles' immunogenicity will be tested in mice and monkeys. In the case of other successful vaccines, such as HBsAg of hepatitis B virus, and L1 capsid protein of human papilloma virus, multimers were up to 1,000-fold more potent than the same weight of monomers. Yet, so far, all HIV vaccines tested in humans have been monomers. If the vaccine elicits a strong immune response in monkeys, they will be challenged with titered stocks of SHIV challenge virus. This is the first example of a gp120-rich particulate vaccine. Through potent carrier and multimer effects, this construct may have a profound effect on HIV immunology. This project is funded by an intramural NIH grant.